An in vitro transcription system which faithfully reflects in vivo transcription has been developed. This project seeks to utilize this system to study the production of adenovirus primary transcripts and the processing and modification of those transcripts into messenger RNA. The major effort is to utilize pulse labeled RNA which has been size fractionated on polyacrylamide gels under denaturing conditions. The RNA that is greater than 28S in size is eluted from the gels and the sequences analyzed by hybridization elution and rehybridization to DNA restriction fragments. Alternative approaches to sequence will utilize the transfer of RNA to filters followed by hybridization with nick-translated DNA fragments. The other is to analyze the fragment selected RNA by oligonucleotide mapping. These studies will be performed as a function of time and the processing of the primary transcripts will be examined. The nature of poly A addition in this in vitro system and the role of poly A addition in the processing of these RNAs will be examined. These studies should greatly enhance our knowledge of the molecular mechanisms involved in the regulation of gene expression in general and the expression of viral genes in particular.